Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
>chr2:72210271+72210368 98bp ACCATCACTGCTTCCGAGA GGCTCTACAGTGGTGCTTCC
Mutant= 107 bp
Wild Type = 98 bp
Wt Sequence: accatcactgcttccgagaaacaaataatgcagagcctgaccagcgcgaggcctGCcgggcacgctgtcgagctgggtggaagcaccactgtagagcc
Mutant Sequence: acgcccagcttatatcgtgaagttagaaatgtagtggtttcaacgttagaaacatgacgccatACcgggcacgctgtcgagctgggtggaagcaccactgtagagcc
609 bp deletion beginning at Chromosome 2 positive strand position 72,371,660 bp CTTTTCTTTGGTGCTAAGGT, and ending after CCTGACCAGCGCGAGGCCTG at 72,372,268 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35176 | Fluorophore-1 | CAG AGC CTG ACC AGC GC | Quencher-1 | WT Probe | ||
35177 | Fluorophore-2 | CAA CGT TAG AAA CAT GAC GCC | Quencher-2 | MUT Probe | ||
35178 | ACC ATC ACT GCT TCC GAG A | Wild type Forward | A | |||
35179 | GGC TCT ACA GTG GTG CTT CC | Common | A | |||
35180 | ACG CCC AGC TTA TAT CGT GA | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35178 | 0.40 uM |
35179 | 0.40 uM |
35180 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |